The regulation of the sulfur amino acid biosynthetic pathway in Cryptococcus neoformans: the relationship of Cys3, Calcineurin, and Gpp2 phosphatases
The regulation of the sulfur amino acid biosynthetic pathway in Cryptococcus neoformans: the relationship of Cys3, Calcineurin, and Gpp2 phosphatases
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DOI：
10.1038/s41598-019-48433-5
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发表时间：
2019-08-15
影响因子：
4.6
通讯作者：
Pascon, Renata C.
中科院分区：
文献类型：
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作者：
de Melo, Amanda Teixeira;Martho, Kevin Felipe;Pascon, Renata C.
隐球菌病是由新生隐球菌属引起的一种真菌病。为了适应和生存在包括动物宿主在内的各种生态位中，这种机会性病原体依赖于其吸收营养物质的能力，如碳、氮、铁、磷、硫和氨基酸。遗传回路在对环境变化的反应中发挥作用，调节基因表达并调整微生物代谢以获得最佳能量利用和生存所需的营养物质。我们研究了含硫氨基酸的生物合成及其对新生葡萄球菌生物学和毒力的影响。CNAG_04798编码一个BZIP蛋白，被注释为Cys3，被认为是一个必需基因。然而，我们证明了CyS3不是必需的，事实上，它的敲除导致了含硫氨基酸的营养缺陷体。Western blotts和荧光显微镜观察表明，GFP-Cys3在富营养液(YEPD)中定位于细胞核；蛋氨酸和半胱氨酸作为唯一氮源(SD-N+Met/Cys)导致细胞核定位减少和蛋白质降解。通过蛋白质组学方法，我们鉴定并证实了Gpp2、Cna1、Cnb1和GFP-Cys3之间的物理相互作用。GFP-Cys3背景中钙调神经磷酸酶和GPP2基因的缺失表明，在YEPD中，钙调神经磷酸酶是维持Cys3高蛋白水平所必需的，而GPP2的缺失导致GFP-Cys3在含硫氨基酸的存在下持续存在。RNAseq对突变体和野生型的整体转录图谱显示，Cys3控制硫氨基酸生物合成的所有分支，硫饥饿导致几条氨基酸生物合成途径的诱导。此外，我们还发现，在梅隆格列氏菌动物模型中，Cys3是毒力所必需的。
Cryptococcosis is a fungal disease caused by C. neoformans. To adapt and survive in diverse ecological niches, including the animal host, this opportunistic pathogen relies on its ability to uptake nutrients, such as carbon, nitrogen, iron, phosphate, sulfur, and amino acids. Genetic circuits play a role in the response to environmental changes, modulating gene expression and adjusting the microbial metabolism to the nutrients available for the best energy usage and survival. We studied the sulfur amino acid biosynthesis and its implications on C. neoformans biology and virulence. CNAG_04798 encodes a BZip protein and was annotated as CYS3, which has been considered an essential gene. However, we demonstrated that CYS3 is not essential, in fact, its knockout led to sulfur amino acids auxotroph. Western blots and fluorescence microscopy indicated that GFP-Cys3, which is expressed from a constitutive promoter, localizes to the nucleus in rich medium (YEPD); the addition of methionine and cysteine as sole nitrogen source (SD-N + Met/Cys) led to reduced nuclear localization and protein degradation. By proteomics, we identified and confirmed physical interaction among Gpp2, Cna1, Cnb1 and GFP-Cys3. Deletion of the calcineurin and GPP2 genes in a GFP-Cys3 background demonstrated that calcineurin is required to maintain Cys3 high protein levels in YEPD and that deletion of GPP2 causes GFP-Cys3 to persist in the presence of sulfur amino acids. Global transcriptional profile of mutant and wild type by RNAseq revealed that Cys3 controls all branches of the sulfur amino acid biosynthesis, and sulfur starvation leads to induction of several amino acid biosynthetic routes. In addition, we found that Cys3 is required for virulence in Galleria mellonella animal model.